WO2018014452A1 - 一种毕赤酵母表达重组蛋白的发酵工艺 - Google Patents

一种毕赤酵母表达重组蛋白的发酵工艺 Download PDF

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WO2018014452A1
WO2018014452A1 PCT/CN2016/102430 CN2016102430W WO2018014452A1 WO 2018014452 A1 WO2018014452 A1 WO 2018014452A1 CN 2016102430 W CN2016102430 W CN 2016102430W WO 2018014452 A1 WO2018014452 A1 WO 2018014452A1
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fermentation
pichia pastoris
stage
culture
recombinant protein
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PCT/CN2016/102430
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杨树林
赵健烽
黄建民
高力虎
杜尔凤
陶海
冯丽萍
季乐
周爱梅
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江苏江山聚源生物技术有限公司
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Priority to KR1020197001822A priority Critical patent/KR20190018724A/ko
Priority to US16/318,549 priority patent/US11104928B2/en
Priority to EP16909379.6A priority patent/EP3473725B1/en
Publication of WO2018014452A1 publication Critical patent/WO2018014452A1/zh

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    • C12P21/00—Preparation of peptides or proteins
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    • C12R2001/84—Pichia

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  • the invention belongs to the technical field of fermentation engineering, and particularly relates to a fermentation process for expressing recombinant protein by Pichia pastoris.
  • the Pichia pastoris expression system is a novel exogenous protein expression system developed in the early 1980s. It has a prokaryotic expression system that is easy to operate, easy to culture, fast growing, and high in expression.
  • the invention has the advantages of low cost and the like, and also has the characteristics of exogenous protein modification such as glycosylation and protein phosphorylation which are not possessed by prokaryotic expression systems.
  • Pichia pastoris expression system has the advantages of Pichia pastoris expression system: (1) having the alcohol oxidase aoxl gene promoter, which is one of the strongest and most stringent promoters; (2) high expression efficiency, and its expression The source protein can account for more than 90% of the total expressed protein, which is beneficial to the separation and purification of the target protein; (3) high-density culture can be achieved in a simple synthetic medium; (4) the expression plasmid can be a single copy at a specific site of the genome. Or multiple copies of the form of stable integration; (5) the yeast can be the sole carbon source of methanol, the medium components do not need to add other organic matter, reducing pollution.
  • the fermentation process of Pichia pastoris developed by Invitrogen can be divided into primary and secondary seed culture, glycerol culture stage, glycerol flow stage and methanol induction stage.
  • the process is currently the production of foreign protein by Pichia pastoris, and the more common fermentation process (Chinese patent) 201010602114.5, 201110327865.5).
  • the purpose of glycerol culture and glycerol flow is to make the cells grow and proliferate rapidly, and achieve the optimal concentration range of high-density fermentation. This stage is very important in industrial production. It needs real-time monitoring, complicated operation and difficult to control. At or below this range, there will be adverse consequences of increased costs and reduced protein production.
  • the object of the present invention is to simplify the fermentation process of the existing Pichia pastoris expressing recombinant protein, and provide a simple process, convenient and feasible operation, large yield of recombinant protein, high purity, and suitable for industrialized large-scale production of Pichia pastoris. Fermentation process of recombinant protein.
  • the glycerin addition amount in the glycerol culture stage is 60-70g/L.
  • the Pichia pastoris of the present invention has been deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on June 29, 2011, and the preservation number is CGMCC No. 5021, and is fully enriched in Chinese Patent 201110327865.5. public.
  • the first-stage seed culture according to the present invention adopts the existing conventional method, specifically, the strain Pichia pastoris is inoculated into the seed culture medium, and cultured at 30 ° C for 24 to 36 hours, to the wet weight of the bacteria. It reaches 20 ⁇ 2g/L.
  • the secondary seed culture of the present invention adopts the existing conventional method, specifically, the first-stage seed liquid is transferred into the fermentation medium, cultured at 30 ° C, adjusted with ammonia water and controlled to have a pH of 5.0, and dissolved oxygen is controlled at 20% to 30%, the wet weight of the cells is 120 ⁇ 10g/L.
  • the specific step of the glycerol culture stage of the present invention is to transfer the second-stage seed liquid into the fermentation medium, add 60-70 g/L glycerin, culture at 30 ° C, adjust with ammonia water and control the pH value of 5.0, ventilation It is 30 m 3 /h, the stirring rate is 300-500 rpm, and when the dissolved oxygen rises abruptly, starving for 1 h causes the glycerin to be depleted.
  • the methanol induction stage of the present invention adopts the existing conventional method, specifically, the temperature of the control induction stage is 30 ° C, the pH value is 5.0, the methanol feed and the dissolved oxygen linkage are set, and the flow starts when the dissolved oxygen is higher than 20%.
  • the present invention has the following advantages:
  • the fermentation process of Pichia pastoris of the present invention eliminates the need to prepare for the addition of sterile glycerin, and simplifies the glycerin sterilization tank, thereby reducing energy, resource loss, and waste of glycerin;
  • the fermentation process of the Pichia pastoris of the invention is simple and easy, no need to monitor the concentration of the bacteria, reduce the probability of error, and is convenient for the production personnel to implement, and is more suitable for large-scale industrial production.
  • the process of the invention is simple, the yield of the target protein is equivalent, the recovery rate is increased to about 70%, and the purity is over 95%, which is more suitable for industrial large-scale production of recombinant human collagen.
  • Fig. 1 is a HPLC detection diagram of the fermentation broth obtained in Comparative Example 1 (a) and Example 2 (b) after separation and purification.
  • the fermentation process for expressing recombinant protein of Pichia pastoris comprises four stages: first stage seed culture, second stage seed culture, glycerol culture stage, and methanol flow addition stage.
  • the existing fermentation process for expressing recombinant protein of Pichia pastoris comprises five stages: first stage seed culture, second stage seed culture, glycerol culture stage, glycerol flow addition stage, and methanol flow addition stage.
  • strains and the respective media used in the examples and comparative examples of the present invention are basically as follows:
  • the first-stage seed medium is BMGY medium, and the composition is:
  • composition of the secondary seed medium is:
  • the fermentation medium includes the following components:
  • the glycerin concentration in the comparative example was 40 g/L, and a glycerin aqueous solution having a mass concentration of 50% was added thereto.
  • the glycerin concentration in the examples was 55 to 75 g/L, and after the end of the glycerin stage, the methanol addition stage was entered, and the medium was not replaced.
  • composition of PTM1 is:
  • a fermentation process for expressing proteins of Pichia pastoris the steps are as follows:
  • the glycerol-preserved strain was inoculated into a shake flask containing 200 mL of seed medium, and cultured at 30 ° C, 250 rpm for 24 h to 36 h, and cultured until the wet weight of the cells reached 20 ⁇ 2 g / L;
  • the first-stage seed liquid was transferred to a 100-liter fermenter containing 60 L fermentation medium, cultured at 30 ° C, adjusted with ammonia water and controlled to pH 5.0, dissolved oxygen was controlled at 20% to 30%, and cultured to wet weight of the cells. Up to 120 ⁇ 10g/L;
  • the secondary seed solution was transferred to a 1000 L fermentor containing 60 L of fermentation medium, 60 g/L of glycerol was added, cultured at 30 ° C, adjusted with ammonia water and controlled to pH 5.0, and the aeration amount was 30 m 3 /h, and the stirring rate was 300 to 500 rpm, when dissolved oxygen rises abruptly, starving for 1 h causes glycerol to be depleted;
  • the temperature of the induction stage is 30 ° C
  • the pH value is controlled at 5.0
  • the methanol feed and dissolved oxygen linkage are set.
  • the dissolved oxygen is higher than 20%
  • the methanol is added, and the dissolved oxygen is lower than 20%.
  • the plateau was reached, and after 120 hours of induction, the cans were centrifuged, and the fermented liquid after centrifugation was centrifuged, and the supernatant after centrifugation was collected for concentration, ultrafiltration and nanofiltration decolorization and desalting to obtain recombinant human collagen.
  • Example 2 differs from Example 1 in that the amount of glycerin added is 65 g/L, and the other steps are the same as in the first embodiment.
  • Example 2 differs from Example 1 in that the amount of glycerin added is 70 g/L, and the other steps are the same as in the first embodiment.
  • This comparative example differs from Example 1 in that the amount of glycerin added is 55 g/L, and other steps are Example 1 is the same.
  • Example 2 differs from Example 1 in that the amount of glycerin added is 75 g/L, and the other steps are the same as in Example 1.
  • the glycerol-preserved strain was inoculated into a shake flask containing 200 mL of seed medium, and cultured at 30 ° C, 250 rpm for 24 h to 36 h, and cultured until the wet weight of the cells reached 20 ⁇ 2 g / L;
  • the first-stage seed liquid was transferred to a 100-liter fermenter containing 60 L fermentation medium, cultured at 30 ° C, adjusted with ammonia water and controlled to pH 5.0, dissolved oxygen was controlled at 20% to 30%, and cultured to wet weight of the cells. Up to 120 ⁇ 10g/L;
  • the secondary seed solution was transferred to a 1000 L fermentor containing 60 L of fermentation medium, 40 g/L of glycerol was added, cultured at 30 ° C, adjusted with ammonia water and controlled to pH 5.0, and the aeration was 30 m 3 /h, and the stirring rate was 300 to 500 rpm, when the dissolved oxygen suddenly rises, the flow of supplemental glycerin is started, the pH value is controlled to 5.0, the dissolved oxygen is controlled to be more than 20%, and when the concentration of the bacteria reaches 160 ⁇ 10 g/L, the flow of glycerol is stopped, and the hunger is 1 h. Depletion of glycerol;
  • the temperature of the induction stage is 30 ° C
  • the pH value is controlled at 5.0
  • the methanol feed and dissolved oxygen linkage are set.
  • the dissolved oxygen is higher than 20%
  • the methanol is added, and the dissolved oxygen is lower than 20%.
  • the plateau was reached, and after 120 hours of induction, the cans were removed; the fermentation broth after induction was centrifuged, and the supernatant after centrifugation was collected and concentrated in turn. Decolorization and desalting by ultrafiltration and nanofiltration to obtain recombinant human collagen.
  • Example 1 is an HPLC detection diagram of the fermentation broth obtained in Comparative Example 1 and Example 2 after separation and purification.
  • the peak in the figure is recombinant human collagen, wherein a is Comparative Example 1, and b is Example 2. It can be seen from the figure that after the separation and purification of the fermentation broth of Comparative Example 1, the protein purity is 91.7%, and the protein purity of the fermentation broth of Example 2 is 96.4% after separation and purification, and the recombinant human collagen obtained by the fermentation process of the present invention.
  • the purity of the protein is superior to the comparative example.
  • the recovery rate of the recombinant human collagen is over 70%, and the purity is over 95%, which is better than the comparative example.
  • the collagen concentration and purity after purification were determined by HPLC, and the protein expression amount was comparable to the comparative example, and the protein yield and purity were higher than the comparative examples.
  • the invention has simple process and is more suitable for industrial large-scale production of recombinant human collagen.

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Abstract

提供一种毕赤酵母表达重组蛋白的发酵工艺，该发酵工艺采用巴斯德毕赤酵母Pichia pastoris为菌种，先进行一级种子培养，至菌浓为20±2g/L时转接二级种子培养，二级种子培养至菌浓为120±10g/L时转接至甘油培养阶段，甘油培养阶段中甘油的添加量为60～70g/L，待溶氧迅速上升到达相对稳定状态时，进入甲醇诱导阶段，诱导120±8h后，发酵结束。

Description

一种毕赤酵母表达重组蛋白的发酵工艺 技术领域
本发明属于发酵工程技术领域，具体涉及一种毕赤酵母表达重组蛋白的发酵工艺。
背景技术
巴斯德毕赤酵母(Pichia pastoris)表达***是上世纪80年代初期发展起来的一种新型的外源蛋白表达***，它既具有原核表达***操作简易、易于培养、生长速度快、表达量高、成本低等优点，还具有原核生物表达***不具有的对外源蛋白的修饰如糖基化、蛋白磷酸化等特点。巴斯德毕赤酵母表达***的优点是：(1)具有醇氧化酶aoxl基因启动子，是目前最强、调控机理最严格的启动子之一；(2)表达效率高，其表达的外源蛋白可占总表达蛋白的90％以上，有利于目的蛋白的分离纯化；(3)在简单合成培养基中可实现高密度培养；(4)表达质粒能在基因组的特定位点以单拷贝或多拷贝的形式稳定整合；(5)该酵母可以甲醇为唯一的碳源，培养基成分无需添加其它有机质，减少污染。
目前常用的表达载体和发酵工艺多是由美国Invitrogen公司构建开发，该***己成功地表达了几百种外源蛋白。Invitrogen公司开发的巴斯德毕赤酵母发酵工艺，其工业化可分为一级、二级种子培养、甘油培养阶段、甘油流加阶段及甲醇诱导阶段。该工艺是目前巴斯德毕赤酵母表达生产外源蛋白，较为通用的发酵工艺(中国专利 201010602114.5，201110327865.5)。甘油培养和甘油流加阶段目的在于使菌体快速生长和增殖，达到高密度发酵最佳的菌体浓度范围，该阶段在工业化生产中十分关键，需实时监控，操作复杂并较难掌控，高于或低于该范围，都将造成成本增加、蛋白产量降低的不良后果。
实际的毕赤酵母表达重组蛋白的工业化生产中，主要操作人员以普通工人为主，过于复杂的操作容易导致较高的出错率，发生技术失误事件进而导致发酵工艺的最终失败。因此，简化毕赤酵母表达重组蛋白的发酵工艺，尤其是简化现有的发酵工艺中复杂的甘油培养和甘油流加阶段的工艺，不仅可以降低生产成本，同时也减少出错的概率，减少因技术失误而导致的发酵失败，能够为重组蛋白的工业化生产带来便利和极大的经济价值。
发明内容
本发明的目的在于简化现有的毕赤酵母表达重组蛋白的发酵工艺，提供一种工艺简单，操作方便可行，重组蛋白产收率大、纯度高，适于工业化大规模生产的毕赤酵母表达重组蛋白的发酵工艺。
本发明的技术方案如下：
一种毕赤酵母表达重组蛋白的发酵工艺，采用巴斯德毕赤酵母Pichia pastoris为菌种，先进行一级种子培养，至菌浓为20±2g/L时转接二级种子培养，二级种子培养至菌浓为120±10g/L时转接至甘油培养阶段，甘油培养阶段中甘油的添加量为60～70g/L，待溶氧迅速上升到达相对稳定状态时，进入甲醇诱导阶段，诱导120±8h后，发酵结束。
本发明所述的巴斯德毕赤酵母Pichia pastoris，已于2011年6月29日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.5021，并在中国专利201110327865.5中充分公开。
本发明所述的一级种子培养采用现有的常规方法，具体为将菌种巴斯德毕赤酵母Pichia pastoris接种至种子培养基中，30℃摇瓶培养24h～36h，至菌体湿重达到20±2g/L。
本发明所述的二级种子培养采用现有的常规方法，具体为将一级种子液全部转接入发酵培养基中，30℃培养，用氨水调节并控制pH值为5.0，溶氧控制在20％～30％，培养至菌体湿重达到120±10g/L。
本发明所述的甘油培养阶段的具体步骤为将二级种子液转入发酵培养基中，添加量60～70g/L的甘油，30℃培养，用氨水调节并控制pH值为5.0，通气量为30m3/h，搅拌速率为300～500rpm，当溶氧陡然上升时，饥饿1h使甘油耗尽。
本发明所述的甲醇诱导阶段采用现有的常规方法，具体为控制诱导阶段温度为30℃，pH值为5.0，设定甲醇补料与溶氧联动，当溶氧高于20％时开始流加甲醇，溶氧低于20％停止流加甲醇，诱导120±8h后，发酵结束，对发酵液进行离心，收集离心后的上清液依次进行浓缩、超滤和纳滤脱色脱盐，得到重组人源胶原蛋白。
与现有技术相比，本发明具有以下优点：
(1)本发明的毕赤酵母的发酵工艺，不再需要准备流加用无菌甘油，精简了甘油灭菌罐，减少了能源、资源的损耗以及甘油的浪费；
(2)本发明甘油消耗完即可进入下一工艺流程，有效解决了毕 赤酵母发酵工艺复杂，蛋白产量不稳定的问题，同时缩短了甘油培养阶段的时间；
(3)本发明的毕赤酵母的发酵工艺，简便易行，无需再监控菌体浓度，减少了出错几率，便于生产人员实施，更适合大规模工业化生产。
与原工艺相比，本发明工艺简单，目的蛋白产率相当，并且回收率提高至70％左右，纯度达95％以上，更适合于工业化大规模生产重组人源胶原蛋白。
附图说明
图1为对比例1(a)和实施例2(b)得到的发酵液经分离纯化后的HPLC检测图。
具体实施方式
本发明的毕赤酵母表达重组蛋白的发酵工艺，包括四个阶段：一级种子培养，二级种子培养，甘油培养阶段，甲醇流加阶段。
现有的毕赤酵母表达重组蛋白的发酵工艺，包括五个阶段：一级种子培养，二级种子培养，甘油培养阶段，甘油流加阶段，甲醇流加阶段。
除特殊说明外，本发明的实施例和对比例中使用的菌种及各培养基基本如下：
1、菌种：巴斯德毕赤酵母Pichia pastoris，保藏编号：CGMCCNo.5021，已于2011年6月29日保藏于中国微生物菌种保藏管理委员会普通微生物中心，并在中国专利201110327865.5中充分公开。
2、培养基：
(1)一级种子培养基为BMGY培养基，组成为：
Figure PCTCN2016102430-appb-000001
(2)二级种子培养基组成为：
Figure PCTCN2016102430-appb-000002
(3)发酵培养基包括以下组分：
Figure PCTCN2016102430-appb-000003
对比例中的甘油浓度为40g/L，流加质量浓度为50％的甘油水溶液。实施例中的甘油浓度为55～75g/L，甘油阶段结束后进入甲醇流加阶段，不再更换培养基。
其中PTM1组成为：
Figure PCTCN2016102430-appb-000004
实施例1
一种毕赤酵母表达蛋白的发酵工艺，步骤如下：
(1)一级种子培养
将甘油保存的菌种接种至含有200mL种子培养基的摇瓶中，30℃，250rpm培养24h～36h，培养至菌体湿重达到20±2g/L；
(2)二级种子培养
将一级种子液全部转接入含有60L发酵培养基的100L发酵罐中，30℃培养，用氨水调节并控制pH值为5.0，溶氧控制在20％～30％，培养至菌体湿重达到120±10g/L；
(3)发酵罐培养和诱导表达
将二级种子液转入含有60L发酵培养基的1000L发酵罐中，添加60g/L的甘油，30℃培养，用氨水调节并控制pH值为5.0，通气量为30m3/h，搅拌速率为300～500rpm，当溶氧陡然上升时，饥饿1h使甘油耗尽；
然后开始甲醇诱导，诱导阶段温度为30℃，pH值控制在5.0，设定甲醇补料与溶氧联动，当溶氧高于20％开始流加甲醇，溶氧低于20％停止流加甲醇，诱导112后达到平台期，诱导120h后出罐，对诱导表达后的发酵液进行离心，收集离心后的上清液依次进行浓缩、超滤和纳滤脱色脱盐，得到重组人源胶原蛋白。
实施例2
本实施例与实施例1不同的是甘油添加量为65g/L，其他步骤与实施例1相同。
实施例3
本实施例与实施例1不同的是甘油添加量为70g/L，其他步骤与实施例1相同。
对比例1
本对比例与实施例1不同的是甘油添加量为55g/L，其他步骤与 实施例1相同。
对比例2
本实施例与实施例1不同的是甘油添加量为75g/L，其他步骤与实施例1相同。
对比例3
(1)一级种子培养
将甘油保存的菌种接种至含有200mL种子培养基的摇瓶中，30℃，250rpm培养24h～36h，培养至菌体湿重达到20±2g/L；
(2)二级种子培养
将一级种子液全部转接入含有60L发酵培养基的100L发酵罐中，30℃培养，用氨水调节并控制pH值为5.0，溶氧控制在20％～30％，培养至菌体湿重达到120±10g/L；
(3)发酵罐培养和诱导表达
将二级种子液转入含有60L发酵培养基的1000L发酵罐中，添加40g/L的甘油，30℃培养，用氨水调节并控制pH值为5.0，通气量为30m3/h，搅拌速率为300～500rpm，当溶氧陡然上升时，开始流加补甘油，pH值控制为5.0，溶氧控制在20％以上，当菌体浓度达到160±10g/L时停止流加甘油，饥饿1h使甘油耗尽；
然后开始甲醇诱导，诱导阶段温度为30℃，pH值控制在5.0，设定甲醇补料与溶氧联动，当溶氧高于20％开始流加甲醇，溶氧低于20％停止流加甲醇，诱导112后达到平台期，诱导120h后出罐；对诱导表达后的发酵液进行离心，收集离心后的上清液依次进行浓缩、 超滤和纳滤脱色脱盐，得到重组人源胶原蛋白。
图1为对比例1和实施例2得到的发酵液经分离纯化后的HPLC检测图，图中峰为重组人源胶原蛋白，其中a为对比例1，b为实施例2。从图中可知，对比例1的发酵液经分离纯化后，蛋白纯度为91.7％，实施例2的发酵液经分离纯化后，蛋白纯度为96.4％，本发明的发酵工艺得到的重组人源胶原蛋白的纯度优于对比例。
对各实施例和对比例中的发酵结果进行检测，分别测定甲醇流加前菌体湿重、胶原蛋白产量、蛋白回收率和蛋白纯度，具体检测结果如表1所示。
表1 各实施例和对比例的发酵结果
  甲醇流加前菌体湿重 胶原蛋白产量 蛋白回收率 蛋白纯度
对比例1 147.8g/L 14.51g/L 67.2％ 91.7％
对比例2 186.2g/L 15.43g/L 67.9％ 93.4％
对比例3 166.7g/L 16.82g/L 62.8％ 92.1％
实施例1 163.3g/L 16.93g/L 70.7％ 95.3％
实施例2 169.4g/L 17.02g/L 73.3％ 96.4％
实施例3 177.8g/L 16.87g/L 72.8％ 95.9％
从表中可以看出，采用本发明发酵工艺，重组人源胶原蛋白的回收率达到70％以上，纯度达到95％以上，优于对比例。利用HPLC测定纯化之后胶原蛋白浓度和纯度，蛋白表达量与对比例相当，蛋白收率和纯度均高于对比例。综上所述，本发明工艺简单，更适合于工业化大规模生产重组人源胶原蛋白。

Claims (6)

  1. 一种毕赤酵母表达重组蛋白的发酵工艺，其特征在于，采用巴斯德毕赤酵母Pichia pastoris为菌种，先进行一级种子培养，至菌浓为20±2g/L时转接二级种子培养，二级种子培养至菌浓为120±10g/L时转接至甘油培养阶段，甘油培养阶段中甘油的添加量为60～70g/L，待溶氧迅速上升到达相对稳定状态时，进入甲醇诱导阶段，诱导120±8h后，发酵结束。
  2. 根据权利要求1所述的毕赤酵母表达重组蛋白的发酵工艺，其特征在于，所述的巴斯德毕赤酵母Pichia pastoris，保藏编号为CGMCC No.5021。
  3. 根据权利要求1所述的毕赤酵母表达重组蛋白的发酵工艺，其特征在于，所述的一级种子培养为将菌种巴斯德毕赤酵母Pichia pastoris接种至种子培养基中，30℃摇瓶培养24h～36h，至菌体湿重达到20±2g/L。
  4. 根据权利要求1所述的毕赤酵母表达重组蛋白的发酵工艺，其特征在于，所述的二级种子培养为将一级种子液全部转接入发酵培养基中，30℃培养，用氨水调节并控制pH值为5.0，溶氧控制在20％～30％，培养至菌体湿重达到120±10g/L。
  5. 根据权利要求1所述的毕赤酵母表达重组蛋白的发酵工艺，其特征在于，所述的甘油培养阶段为将二级种子液转入发酵培养基中，添加60～70g/L的甘油，30℃培养，用氨水调节并控制pH值为5.0，通气量为30m3/h，搅拌速率为300～500rpm，当溶氧陡然上升时，饥 饿1h使甘油耗尽。
  6. 根据权利要求1所述的毕赤酵母表达重组蛋白的发酵工艺，其特征在于，所述的甲醇诱导阶段为控制诱导阶段温度为30℃，pH值为5.0，设定甲醇补料与溶氧联动，当溶氧高于20％时开始流加甲醇，溶氧低于20％停止流加甲醇，诱导120±8h后，发酵结束，对发酵液进行离心，收集离心后的上清液依次进行浓缩、超滤和纳滤脱色脱盐，得到重组人源胶原蛋白。
PCT/CN2016/102430 2016-07-22 2016-10-18 一种毕赤酵母表达重组蛋白的发酵工艺 WO2018014452A1 (zh)

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